Despite the use of a generally safe vaccine for 70 years and effective antibiotics for 40 years, tuberculosis still kills approximately 3 million people annually. To develop new methods of diagnosis and therapy, it is necessary to find new targets, i.e., genes and proteins, in the causative organism, Mycobacterium tuberculosis. An understanding of mycobacterial gene expression is expected to provide much assistance in achieving this goal. In recent years, the investigator's laboratory has studied general and specific mechanisms of gene regulation, i.e., the structure and function of the basic mycobacterial transcriptional apparatus and the control of iron acquisition. Much has been learned regarding these regulatory systems in pathogenic and non-pathogenic mycobacteria, both biochemically and in vitro. Information gained in these initial experiments is now being used to study the in vivo regulation of M. tuberculosis genes and their functions during the infectious process. The aims of this new grant proposal are: 1) identification of M. tuberculosis genes that are specifically induced, in vivo. An IVET promoter trap system has been developed. Several genes have been isolated thus far, and the search for new candidate genes will be continued; 2) studying the expression of M. tuberculosis genes in vivo. A sensitive and accurate real time RT-PCR method has been developed to quantitate the in vivo expression of M. tuberculosis genes that have been and will be identified in the experiments described in first aim. In addition, they can also measure the expression of other M. tuberculosis genes that they believe may be induced (or repressed) upon infection of macrophages. Several genes showing differential regulation in macrophages have already been identified and they will study the mechanisms by which they are induced or repressed after infection. Since some of these genes are iron regulated, they will also manipulate iron levels in macrophages to measure the affects on their expression; 3) elucidating the role of in vivo-induced M. tuberculosis genes in virulence. In the course of these studies, they expect to identify many genes that are induced in vivo. To determine their role in virulence, they will inactivate these genes in M. tuberculosis and will measure the survival and growth of the mutants in various macrophages and in mice.